A major unsolved problem of cell biology and biochemistry is the elucidation of the mechanisms of intracellular phospholipid transport and assembly into the organelles of eukaryotic cells. While considerable information has accrued to define the mechanisms of intracellular protein transport, little detailed information is available to describe the lipid transport at the molecular level. Lipid transport is essential for all cell growth, development, replication and homeostasis. The long term goals of this project are to define intracellular lipid transport at the molecular level using biochemical and genetic approaches in the yeast Saccharomyces cerevisiae. This proposal will extend recent advances made in identifying specific genes involved in controlling the movement of phosphatidylserine and phosphatidylethanolamine among different organelles. In Specific Aim 1 we will address the role of protein ubiquitination in regulating phosphatidylserine transport between the endoplasmic reticulum and mitochondria. We will use biochemical and genetic manipulations in vitro and in vivo to regulate protein ubiquitination, and perform proteomic analysis to identify novel substrates for ubiquitination. In Specific Aim 2 we will examine the role of specific lipids, proteins, and protein subdomains in promoting phosphatidylserine transport between the endoplasmic reticulum and Golgi. We will use permeabilized cells and isolated organelles derived from wild type and mutant strains to identify specific lipid and protein components required for this phosphatidylserine transport. Specific Aim 3 will focus upon the genetics and biochemistry of phosphatidylethanolamine export from the Golgi to the endoplasmic reticulum. We will characterize the transport defects caused by multiple genes implicated in the process by previous genetic screens and develop reconstitution methods for the process in vitro. Our combined genetic and biochemical approach will provide new molecular and mechanistic information about the phospholipid transport processes in eukaryotic cells and provide new insights into the regulation of membrane biogenesis.